Malaria Immunology and Pathogenesis The goals of the malaria pathogenesis/immunology program are: 1) To understand how Malian children with hemoglobin S, alpha-thalassemia, G6PD deficiency, and ABO blood group polymorphisms are protected from Plasmodium falciparum malaria.2) To develop a profile of the acquisition of malaria immunity in Malian children.3) To examine various aspects of pathogenesis due to malaria in Malian children and adults.4) To characterize the pharmacokinetics of artemisinin (ART) in this population. These goals are being accomplished through a 4-year longitudinal study of 1500 children ranging in age from 6 months to 18 years living in 3 villages in Mali (the Kenieroba study). One major effort is to determine the relative protection against malaria conferred by different red blood cell (RBC) polymorphisms, and all the enrolled children have been typed for a series of these polymorphisms. In the past 4 years, we have diagnosed and treated 4207 episodes of uncomplicated and severe falciparum malaria. Analysis of our data shows that sickle cell trait (HbAS) confers significantly greater protection against malaria than other RBC polymorphisms, and that age, a surrogate of naturally acquired immunity, is also associated with reduced malaria incidence. We are now investigating the mechanistic basis for this protection in a number of ways: 1)We identified a sub-cohort of children, selecting those with sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed for development of humoral and cellular responses to blood-stage malaria antigens and we have found that antibodies to a variety of merozoite proteins are lower in HbAS children.2) We have developed a plasma reactivity assay using flow cytometry to measure antibody binding to infected red cells and shown that HbAS children do not display increased reactivity to the red cell surface. 3) We have followed 300 HbA, HbS, and HbC children weekly for parasitemia and malaria symptoms to compare asymptomatic parasite infections. 4) We are testing the effects of HbAS and naturally-acquired IgG on the binding of parasitized RBCs to microvascular endothelial cells (MVECs), a critical event in malaria pathogenesis.5) We are determining whether sera from HbAS children display reactivity to selected PfEMP1 molecules or domains. Some children with malaria display severe pathology, but it has been unclear as to whether certain P. falciparum strains elicit more pathology or whether aspects of the host genetics or responses contribute to susceptibility. We are conducting a series of studies related to pathology during malaria including:1) Reactivity of sera from adult males and females from Mali to various domains of a particular PfEMP1 protein, VAR2CSA, which has been implicated in pregnancy associated malaria. 2) Finding that uric acid levels increase during a malaria infection and correlate with disease severity.3)Obtaining evidence that uric acid can promote endothelial cell pathology.4)Demonstrating that the concentration of microparticles in the plasma is elevated in children with malaria.5) Collaborating with Dr. Michael Walther(LMIV) in examining the role of Tregulatory cells in severe malaria using data and peripheral blood samples from our cohort. We have continued a major initiative begun in 2010 to assess the in vitro and in vivo responses of Malian children to artesunate (ART), an effort prompted by reports from Asia of slow clearance of P. falciparum infections. In the past two years we have determined the clearance times of parasites in 215 Malian children with uncomplicated malaria by obtaining parasite counts of blood smears every 6 hours after treatment until clearance is complete. Fortunately, parasites from all children cleared quickly, indicating that the ART resistance phenotype is not present in our study population. Importantly, we have also observed that older children exhibit more rapid clearance times than younger children. We are testing the hypothesis that plasma IgG accelerates the parasite clearance rate in response to ART. Our studies supported the oral or poster presentation of 10 abstracts to the 2011 ASTMH meeting. All members of Dr. Fairhursts laboratory, many members of Dr. Longs laboratory, Dr. Michael Walther (LMIV) and several other LMVR trainees are presently using data, parasites, and/or patient blood samples from our Kenieroba protocol. Mosquito Vectors We continued to address a major issue in vector biology related to the dry season ecology of mosquitoes in the Sahel, viz., does An. gambiae persist during the dry season by aestivation? Continuous collection of mosquitoes throughout four years in a Malian village revealed that (i) mosquitoes are highly clustered during the dry season; (ii) these hot zones are located in the periphery of the village unlike the situation in the wet season; and (iii) mosquitoes shelter outside houses. We evaluated a novel approach to find aestivation shelters by using a trained sniffing dog to locate mosquitoes tagged with a foreign scent. During the dry season, we find 4 mosquitoes/day and we can scent-tag and release them. We have obtained promising results in a preliminary experiment with a trained dog and will move this to the field. We have also continued our analysis of physiological and behavioral changes that underlie aestivation in mosquitoes. To understand the mechanisms facilitating aestivation, mosquitoes were subjected to a sequence of experiments to measure their individual metabolism, flight activity, body size, egg development and oviposition, feeding response, nutritional reserves, and cuticular hydrocarbons. Sand fly / Leishmaniasis Program The LMVR program on leishmaniasis and its sandfly vectors is designed to describe the unexamined epidemiology of cutaneous leishmaniasis (CL) in Mali. Phlebotomus duboscqi is the principal vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL) and is the suspected vector in Mali. We conducted a three-year study in two neighboring villages, Kemena and Sougoula, in Central Mali, an area with a leishmanin skin test positivity of up to 45%. Initially we evaluated the overall diversity of sand flies and found that P. duboscqi represented 99% of the collected Phlebotomus species, and was the primary sand fly inside dwellings. The seasonality and infection prevalence of P. duboscqi was monitored over two consecutive years. Using a quasi-Poisson model we observed a significant annual, seasonal, and village effect on the number of collected P. duboscqi. The infection status of pooled P. duboscqi females was determined by PCR, and the infection prevalence was 2.7%, incriminating P. duboscqi as the vector of CL. Based on PCR, L. major was identified as the only species found in the flies. Importantly, there is no previous data characterizing cellular immune responses to sand fly salivary proteins in leishmania endemic areas. In animal models sand fly salivary proteins which induce a Th1 response or delayed-type hypersensitivity (DTH) confer protection against leishmaniasis. DTH to sand fly bites is also observed in human volunteers, and we are investigating the duration and nature (Th1 versus Th2) of cell-mediated immunity to sand fly salivary proteins in the two villages. Tick-borne relapsing fever In collaboration with RML, we have continued a study of tick-borne relapsing fever (TBRF) in Mali. TBRF is caused by spirochetes maintained in enzootic foci that involve various small mammals and argasid ticks. Through multiple field expeditions, we investigated 20 villages in Mali. By inspection of small rodents and ticks (Ornithodoros sonrai), we found 11.3% of rodents and 18% of ticks were seropositive for TBRF, with a smaller percentage actively infected.